Lane 1: Wild-type HAP1 cell lysate (40 µg)
Lane 2: FER knockout HAP1 cell lysate (40 µg)
Lane 3: HeLa cell lysate (20 µg)
Lane 4: Jurkat cell lysate (20 µg)
Lanes 1 - 4: Merged signal (red and green). Green - ab52479 observed at 100 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab52479 was shown to specifically react with FER when FER knockout samples were used. Wild-type and FER knockout samples were subjected to SDS-PAGE. Ab52479 and ab8245 (loading control to GAPDH) were diluted at 1/5000 and 1:10,000 dilution respectively and incubated overnight at 4C. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1:10,000 dilution for 1 hour at room temperature before imaging.

Overlay histogram showing Jurkat cells stained with ab52479 (red line). The cells were fixed with 4% paraformaldehyde (10 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab52479, 1/100 dilution) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1µg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line). Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter. This antibody gave a positive signal in Jurkat cells fixed with 80% methanol (5 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.

All lanes : Anti-FER antibody [EP1842Y] (ab52479) at 1/1000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : FER knockout HeLa cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 93 kDa
Observed band size: 100 kDa why is the actual band size different from the predicted?

Lanes 1 - 2: Merged signal (red and green). Green - ab52479 observed at 100 kDa. Red - loading control ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37 kDa.

ab52479 was shown to react with FER in wild-type HeLa cells in Western blot with loss of signal observed in FER knockout cell line ab265226 (FER knockout cell lysate ab257950). Wild-type and FER knockout HeLa cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3 % milk in TBS-T (0.1 % Tween^®) before incubation with ab52479 and ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4 °C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 h at room temperature before imaging.

Anti-FER antibody [EP1842Y] (ab52479) at 1/5000 dilution + Jurkat cell lysate at 10 µg

Secondary
Goat anti-rabbit HRP labeled at 1/2000 dilution

Predicted band size: 93 kDa
Observed band size: 93 kDa

FER was immunoprecipitated using 0.5mg Jurkat whole cell extract, 5µg of Rabbit polyclonal to FER and 50µl of protein G magnetic beads (+). No antibody was added to the control (-).
The antibody was incubated under agitation with Protein G beads for 10min, Jurkat whole cell extract lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation.
Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70^oC; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab52479.
Secondary: Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697).
Band: 93kDa; FER